Development of a Phage Cocktail to Target Salmonella Strains Associated with Swine

Infections caused by multidrug resistant Salmonella strains are problematic in swine and are entering human food chains. Bacteriophages (phages) could be used to complement or replace antibiotics to reduce infection within swine. Here, we extensively characterised six broad host range lytic Salmonella phages, with the aim of developing a phage cocktail to prevent or treat infection. Intriguingly, the phages tested differed by one to five single nucleotide polymorphisms. However, there were clear phenotypic differences between them, especially in their heat and pH sensitivity. In vitro killing assays were conducted to determine the efficacy of phages alone and when combined, and three cocktails reduced bacterial numbers by ~2 × 103 CFU/mL within two hours. These cocktails were tested in larvae challenge studies, and prophylactic treatment with phage cocktail SPFM10-SPFM14 was the most efficient. Phage treatment improved larvae survival to 90% after 72 h versus 3% in the infected untreated group. In 65% of the phage-treated larvae, Salmonella counts were below the detection limit, whereas it was isolated from 100% of the infected, untreated larvae group. This study demonstrates that phages effectively reduce Salmonella colonisation in larvae, which supports their ability to similarly protect swine.

Comparative genomics of Acinetobacter baumannii and therapeutic bacteriophages from a patient undergoing phage therapy

In 2016, a 68-year-old patient with a disseminated multi-drug resistant Acinetobacter baumannii infection was treated using lytic bacteriophages in one of the first modern human clinical uses of phage therapy in the United States. Due to the emergency nature of the treatment there was little time to thoroughly characterize the phages used in this intervention or the pathogen itself. Here we report the genomes of the nine phages used for treatment and three strains of A. baumannii isolated prior to and during treatment. The eight phages used in the initial treatment were found to be a group of closely related T4-like myophages; the ninth phage, AbTP3Φ1, was found to be an unrelated Fri1-like podophage. Analysis of 19 A. baumannii isolates collected before and during phage treatment showed that resistance to the T4-like phages appeared as early as two days following the start of treatment. Three A. baumannii strains (TP1, TP2 and TP3) collected before and during treatment were sequenced to closure, and all contained a 3.9 Mb chromosome of sequence type 570 with a KL116 capsule locus and identical 8.7 kb plasmids. Phage-insensitive mutants of A. baumannii strain TP1 were generated in vitro and the majority of identified mutations were located in the bacterial capsule locus. The presence of the same mutation in both the in vitro mutants and in phage-insensitive isolates TP2 and TP3, which evolved in vivo during phage treatment, indicate that in vitro investigations can produce results that are relevant and predictive for the in vivo environment.

Mycobacteriophages to Treat Tuberculosis: Dream or Delusion?

Rates of antimicrobial resistance are increasing globally while the pipeline of new antibiotics is drying up, putting patients with disease caused by drug-resistant bacteria at increased risk of complications and death. The growing costs for diagnosis and management of drug resistance threaten tuberculosis control where the disease is endemic and resources limited. Bacteriophages are viruses that attack bacteria. Phage preparations served as anti-infective agents long before antibiotics were discovered. Though small in size, phages are the most abundant and diverse biological entity on earth. Phages have co-evolved with their hosts and possess all the tools needed to infect and kill bacteria, independent of drug resistance. Modern biotechnology has improved our understanding of the biology of phages and their possible uses. Phage preparations are available to treat meat, fruit, vegetables, and dairy products against parasites or to prevent contamination with human pathogens, such as <i>Listeria monocytogenes, Escherichia coli</i>, or <i>Staphylococcus aureus</i>. Such phage-treated products are considered fit for human consumption. A number of recent case reports describe in great detail the successful treatment of highly drug-resistant infections with individualized phage preparations. Formal clinical trials with standardized products are slowly emerging. With its highly conserved genome and relative paucity of natural phage defence mechanisms <i>Mycobacterium tuberculosis</i> appears to be a suitable target for phage treatment. A phage cocktail with diverse and strictly lytic phages that kill all lineages of <i>M. tuberculosis,</i> and can be propagated on <i>Mycobacterium smegmatis</i>, has been assembled and is available for the evaluation of optimal dosage and suitable routes of administration for tuberculosis in humans. Phage treatment can be expected to be safe and active on extracellular organisms, but phage penetration to intracellular and granulomatous environments as well as synergistic effects with antibiotics are important questions to address during further evaluation.

Phage Therapy in the 21st Century: Is There Modern, Clinical Evidence of Phage-Mediated Efficacy?

Many bacteriophages are obligate killers of bacteria. That this property could be medically useful was first recognized over one hundred years ago, with 2021 being the 100-year anniversary of the first clinical phage therapy publication. Here we consider modern use of phages in clinical settings. Our aim is to answer one question: do phages serve as effective anti-bacterial infection agents when used clinically? An important emphasis of our analyses is on whether phage therapy-associated anti-bacterial infection efficacy can be reasonably distinguished from that associated with often coadministered antibiotics. We find that about half of 70 human phage treatment reports—published in English thus far in the 2000s—are suggestive of phage-mediated anti-bacterial infection efficacy. Two of these are randomized, double-blinded, infection-treatment studies while 14 of those studies, in our opinion, provide superior evidence of a phage role in observed treatment successes. Roughly three-quarters of these potentially phage-mediated outcomes are based on microbiological as well as clinical results, with the rest based on clinical success. Since many of these phage treatments are of infections for which antibiotic therapy had not been successful, their collective effectiveness is suggestive of a valid utility in employing phages to treat otherwise difficult-to-cure bacterial infections.

Evaluation of a pre-harvest bacteriophage therapy for control of Salmonella within bovine peripheral lymph nodes

A series of proof of concept studies were developed to determine if a commercial bacteriophage (phage) cocktail could be utilized for the mitigation of Salmonella in bovine peripheral lymph nodes (LN). The first objective sought to determine if exogenous phage could be isolated from the LN following administration. If successful, the second objective sought to determine if once in the LN, could the phage effectively reduce Salmonella . Salmonella Montevideo was inoculated intradermally in multiple sites and administrations, later followed by delivery of the phage cocktail subcutaneously in two injections around each of the right and left prescapular and subiliac LN. At the conclusion of each study, animals were euthanized and the popliteal and above LN examined. The first study was successful, in that transmission electron microscopy revealed the presence of phage in the LN of the treated cattle, that were identical to the strains in the cocktail. Concentrations of phage were increased ( P &lt; 0.01) in the pre-scapular and subiliac LN in the phage-treated versus control cattle. Subsequent studies modified the protocols to increase Salmonella and phage concentrations within the LN. Overall concentrations of Salmonella were increased in the LN compared to the first study and phage treatment decreased ( P &lt; 0.01) Salmonella in the some of the LN. Phage concentrations were numerically ( P = 0.12), but not statistically, increased in the treated cattle. The final study was modified, hypothesizing that a 48h post-mortem period prior to LN removal would facilitate phage/ Salmonella interaction, however, there were no differences ( P &gt; 0.10) in Salmonella concentrations among treatments. Results demonstrated that Salmonella- specific phages administered to live cattle can translocate to the LN, however once in the LN they had limited to no effect on Salmonella within these nodes.

Phage Treatment Trial to Eradicate LA-MRSA from Healthy Carrier Pigs

The increase of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) causes a threat to human health. LA-MRSA can be transmitted from animals to animal caretakers, which may further spread MRSA to communities and health care facilities. The objective of this work was to study the efficacy of phage treatment in the eradication of LA-MRSA from healthy carrier pigs. A total of 19 MRSA -positive weanling pigs were assigned to a test (n = 10) and a control group (n = 9). A phage cocktail containing three Staphylococcus phages, or a control buffer was administered to the nares and skin of the pigs three times every two days, after which the phage and MRSA levels in nasal and skin swab samples were monitored for a three-week period. The sensitivity of the strains isolated during the follow-up period to the phage cocktail and each phage individually was analyzed and the pig sera were tested for antibodies against the phages used in the cocktail. The phage treatment did not cause any side effects to the pigs. Phages were found in the skin and nasal samples on the days following the phage applications, but there was no reduction in the MRSA levels in the sampled animals. Phage-resistant strains or phage-specific antibodies were not detected during the experiment. The MRSA load in these healthy carrier animals was only 10–100 CFU/swab or nasal sample, which was likely below the replication threshold of phages. The effectiveness of phage treatment to eradicate MRSA from the pigs could thus not be (reliably) determined.

Prokaryotic viruses impact functional microorganisms in nutrient removal and carbon cycle in wastewater treatment plants

As one of the largest biotechnological applications, activated sludge (AS) systems in wastewater treatment plants (WWTPs) harbor enormous viruses, with 10-1,000-fold higher concentrations than in natural environments. However, the compositional variation and host-connections of AS viruses remain poorly explored. Here, we report a catalogue of ~50,000 prokaryotic viruses from six WWTPs, increasing the number of described viral species of AS by 23-fold, and showing the very high viral diversity which is largely unknown (98.4-99.6% of total viral contigs). Most viral genera are represented in more than one AS system with 53 identified across all. Viral infection widely spans 8 archaeal and 58 bacterial phyla, linking viruses with aerobic/anaerobic heterotrophs, and other functional microorganisms controlling nitrogen/phosphorous removal. Notably, Mycobacterium, notorious for causing AS foaming, is associated with 402 viral genera. Our findings expand the current AS virus catalogue and provide reference for the phage treatment to control undesired microorganisms in WWTPs.

Bacteriophage treatment before chemical disinfection can enhance removal of plastic surface-associated Pseudomonas aeruginosa

Opportunistic pathogens can linger on surfaces in hospital and building plumbing environments, leading to infections in at-risk populations. Further, biofilm-associated bacteria are protected from removal and inactivation protocols, such as disinfection. Bacteriophages show promise as tools to treat antibiotic resistant infections. As such, phages may also be useful in environmental applications to prevent newly acquired infections. In the current study, the potential of synergies between bacteriophage and chemical disinfection of the opportunistic pathogen Pseudomonas aeruginosa was assessed under various conditions. Specifically, surface-associated P. aeruginosa was treated with various concentrations of phages (P1 or JG004), chemical disinfectant (sodium hypochlorite or benzalkonium chloride), or combined sequential treatments under three distinct attachment models (spot inoculations, dry biofilms, and wet biofilms). Phages were very effective at removing bacteria in spot inoculation (>3.2 log 10 removal) and wet biofilms (up to 2.6 log 10 removal), while phages prevented regrowth of dry biofilms in the application time. In addition, phage treatment followed by chemical disinfection inactivated more P. aeruginosa under wet biofilm conditions better than either treatment alone. This effect was hindered when chemical disinfection was applied first, followed by phage treatment, suggesting additive benefits of combination treatments are lost when phage is applied last. Further, we confirm prior evidence of greater phage tolerance to benzalkonium chloride relative to sodium hypochlorite, informing choices for combination phage-disinfectant approaches. Overall, this paper further supports the potential of using combination phage and chemical disinfectant treatments to improve inactivation of surface-associated P. aeruginosa . Importance Phages are already utilized in the healthcare industry to treat antibiotic resistant infections, such as on implant-associated biofilms and in compassionate care cases. Phage treatment could also be a promising new tool to control pathogens in the built environment, preventing infections from occurring. This study shows that phage can be combined effectively with chemical disinfectants to improve removal of wet biofilms and bacteria spotted onto surfaces while preventing regrowth in dry biofilms. This has the potential to improve pathogen containment within the built environment and drinking water infrastructure to prevent infections of opportunistic pathogens.

Emerging Phage Resistance in Pseudomonas aeruginosa PAO1 Is Accompanied by an Enhanced Heterogeneity and Reduced Virulence

Bacterial surface structures of a proteinic nature and glycoconjugates contribute to biofilm formation and provide shields to host defense mechanisms (e.g., the complement system and phagocytosis). A loss or alteration of these molecules, leading to phage resistance, could result in fewer virulent bacteria. In this study, we evaluate the biology and phenotype changes in Pseudomonas aeruginosa PAO1 phage-resistant clones, which emerge in phage-treated biofilms. We characterize these clones for phage-typing patterns, antibiotic resistance, biofilm formation, pathogenicity, and interactions with the innate immune system. Another important question that we address is whether phage-resistant mutants are also generated incidentally, despite the phage treatment-selective pressure, as the natural adaptation of the living biofilm population. It is found that the application of different phages targeting a particular receptor selects similar phage resistance patterns. Nevertheless, this results in a dramatic increase in the population heterogeneity, giving over a dozen phage-typing patterns, compared to one of the untreated PAO1 sessile forms. We also confirm the hypothesis that “phage-resistant bacteria are more susceptible to antibiotics and host-clearance mechanisms by the immune system”. These findings support phage application in therapy, although the overall statement that phage treatment selects the less virulent bacterial population should be further verified using a bigger collection of clinical strains.

Efficacy of phage therapy in pigs: systematic review and meta-analysis

Limits on the use and efficacy of various antibiotics coupled with negative consumer perception of the practice have together spurred substantial research into compounds that could reduce the use antibiotics to control bacterial diseases in pigs. Bacteriophages are often among such potential compounds, and various groups have examined the efficacy of bacteriophages or bacteriophage products in limiting transmission or colonization of targeted bacteria. The study presented here provides a systematic review of such studies followed by a meta-analysis of aggregated data produced by each study. The data set was limited to inputs (n = 19; 576 total observations) from studies where: 1) live pigs were inoculated with a known quantity of challenge bacteria; 2) challenged animals were treated with a known quantity of phages; 3) concentrations of the challenge bacteria were measured in different tissues/fluids following phage treatment; and 4) SD (or SE to allow calculation of SD) was reported. Concentrations of challenge bacteria were significantly lower in phage-treated pigs versus challenged but untreated pigs (P &lt; 0.0001; effect size = −1.06 1log10 colony-forming units [CFU]/g). The effect size of phage treatment was significantly greater (P &lt; 0.05) in samples collected 48 to 96 h following phage treatment versus those collected ≤ 24 h following phage treatment. Likewise, effect size of phage treatment was significantly greater in piglets versus market-weight pigs. Across observations, phage treatment effect sizes were greatest (P &lt; 0.01) in fecal samples versus ileal or cecal samples. Taken together, these data indicate that phage treatment can significantly reduce the concentrations of targeted bacteria in pigs; scenarios exist, however, where phage treatment could predictably be more or less effective.